Endo-xylanases are enzymes that randomly cleave the .beta.(1-4) linkages between xylose residues making up the backbone of xylans, a prevalent form of hemicellulose found predominantly in plant primary and secondary cell walls. If this complex plant cell wall polysaccharide is hydrolyzed with xylanases, it can be exploited as a rich source of carbon and energy for the production of livestock and microorganisms. Enzymatic disruption of plant cell walls also increases the efficiency of a number of industrial processes such as juice extraction, retting of flax fibers and pulp production. As discussed in greater detail herein, it will be appreciated that plant cell walls are highly variable structures containing several forms of hemicellulose. Thus, the need exists to identify and produce novel xylanases that are efficient at degrading this complex polysaccharide.
The plant cell wall is a highly variable, complex and resilient structure encasing essentially every cell of higher plants. It represents a rich store of carbon and energy for herbivores as well as an important renewable resource utilized by the pulp and paper, lumber, food, and pharmaceutical industries. The plant cell wall consists largely of polysaccharides and contains lesser amounts of lignin (phenolic esters) and protein. The primary polysaccharide components of plant cell walls are cellulose (a hydrogen-bonded .beta.(1-4)-linked D-glucan), hemicellulose, and pectin (McNeil et al., 1984). Fibrils of cellulose embedded in a matrix of pectin, hemicellulose (comprising various .beta.-xylan polymers), phenolic esters and protein produce a protective structure resistant to dehydration and penetration by phytopathogens through mechanical and enzymatic mechanisms.
Hemicellulose, the second most prevalent polysaccharide in many plant cell walls is composed mainly of xyloglucan or xylan polymers. Xyloglucans consist of a backbone of .beta.-4-linked-D-glucosyl residues substituted with .alpha.-linked D-xylosyl side chains, some of which are extended by fucose, galactose or arabinose residues (McNeil et al., 1984). Xylans have a backbone structure of .beta.(1-4)-linked xylose residues. The structure of xylan is complicated by the attachment of various side chains (e.g., acetic acid, arabinose, coumaric acid, ferulic acid, glucuronic acid, 4-O-methylglucuronic acid) to the xylose residues (McNeil et al., 1984). The strands of hemicellulose are hydrogen bonded to cellulose fibrils to form a strong interconnected lattice.
Cell wall composition varies with plant species, variety, tissue type, growth conditions, and age. Differences in cell wall composition have been reported between dicotyledonous and monocotyledonous plants (Chesson et al., 1995). The primary cell walls of all dicots and many monocots contain greater amounts of xyloglucan than arabinoxylan. In contrast, plants belonging to the family Gramineae (e.g., grasses and cereal) have primary walls in which only cellulose is more abundant than arabinoxylan. Higher pectin concentrations are found in the exterior wall or middle lamellae than in the primary or secondary cells walls. Finally, as cells age, cell walls may become more lignified and resistant to microbial attack.
The complexity of the plant cell wall is related not only to compositional variation but also to the high degree of interaction between constituent cellulose, hemicellulose and pectin molecules. Dual intermeshing networks of polysaccharides, comprising cellulose fibrils crosslinked with hemicellulose and pectic polysaccharides linked by calcium bridges, not only produce a resilient primary cell wall but are of direct relevance to enzymatic degradation (Chesson et al., 1995).
Digestion of the plant cell wall is further complicated by the structure of polysaccharides. Cellulose is a simple unsubstituted polymer of .beta.(1-4)-linked glucose and requires an endoglucanase and cellobiose for complete degradation. In comparison, highly substituted arabinoxylan requires up to seven different enzymes for complete degradation. An endo-xylanase randomly cleaves the xylan backbone into xylooligosaccharides which are subsequently degraded to xylose by a xylosidase. Substituents are cleaved from the xylan backbone with arabinofuranidase, acetylxylan esterase and .alpha.-glucuronidase. Ferulic and p-coumaric acid crosslinks are degraded by feruloyl and p-coumaryl esterases. If complete degradation of the arabinoxylan is not required, fewer enzymes may be needed. Liquefaction of arabinoxylan requires only the shortening of the xylan polymers. Consequently, this objective may be achieved by the production of xylooligosaccharides through the action of a single endo-xylanase. The choice of enzymes is dependent upon the substrate to be degraded.
The known applications of xylanases are numerous. For instance, the treatment of forages with xylanases (along with cellulases) to increase the rate of acid production, thus ensuring better quality silage and improvement in the subsequent rate of plant cell wall digestion by ruminants has been described. Xylanases can be used to treat rye, and other cereals with a high arabinoxylan content to improve the digestibility of cereal by poultry and swine. Xylanases can be used in bioconversion involving the hydrolysis of xylan to xylooligosaccharides and xylose which may serve as growth substrates for microorganisms. This could involve simultaneous saccharification and fermentation. Xylanases can be used in biopulping to treat cellulose pulps to remove xylan impurities or to produce pulps with different characteristics. In some cases they can be applied to reduce the amount of chlorine needed to bleach the pulp and reduce the energy needed for refining pulp. Further, xylanases are useful in the retting of flax fibers, the clarification of fruit juices, the preparation of dextrans for use as food thickeners and the production of fluids and juices from plant materials.
Some characteristics of an endo-xylanase from N. patriciarum strain 27 (from the Agriculture and Agri-Food Canada Lethbridge culture collection) have been reported previously (Tamblyn Lee et al., 1993). Tamblyn Lee et al. described the isolation of a 6.5 kb EcoRI fragment containing a gene encoding an endo-xylanase. The N. patriciarum strain 27 was not disclosed or made publicly available. The location of the xylanase gene was narrowed down to a 3.6-kb EcoRI SaII fragment. Expression of the endo-xylanase gene in E. coli produced at least three proteins (51, 58 and 68 kDa) having xylanase activity. This study did not fully characterize the N. patriciarum strain 27 endo-xylanase gene. No attempt was made to determine the nucleotide sequence of the gene. Nucleotide sequence data is required to create an efficient fusion construct between the endo-xylanase gene and the sequences of a heterologous expression system. Without this information, the large DNA fragments of Tamblyn Lee et al. would not be useful for the construction of a functional gene fusion. This effort would be hampered by a lack of detailed information about the structure of the gene and the location of useful restriction sites. The large DNA fragments identified by Tamblyn Lee et al. are not useful for commercial enzyme production. Specifically, if these large DNA fragments were cloned into efficient expression systems, translation of the resulting transcripts transcribed from a strong heterologous promoter would not be possible as transcription would be terminated at one of the multiple stop codons found in AT rich sequences upstream from the endo-xylanase gene. Further characterization, isolation and nucleotide sequencing of the N. patriciarum strain 27 endo-xylanase gene would be required if it were to be of commercial importance.
In light of the many industrial applications for xylanases, the need for new xylanases is apparent. Accordingly, it is of great importance to obtain genes encoding xylan-degrading enzymes from novel sources which may be brought to expression in other, high-producing microbial or eukaryotic expression systems.